Martina Volarevic

Positional effects and strand preference of RNA interference against hepatitis C virus target sequences

Summary.  The hepatitis C virus (HCV) 3′‐untranslated region (UTR) and negative‐strand RNA sequences contribute cis‐acting functions essential to viral RNA replication. Although efficient suppression of HCV replicon RNA in cell culture has been demonstrated with small interfering RNAs (siRNAs) directed against various sequences in the 5′ UTR and coding regions, data regarding siRNA targeting of the 3′ UTR have been lacking. Furthermore, it has not been definitively shown whether the active constructs, identified to date, exert their effect exclusively via suppression of the replicon positive strand, negative strand or some combination of both strands. In the present study, we assayed inhibitory activity of various siRNAs targeting the 3′ UTR by transient transfection in a subgenomic replicon cell culture model. A survey of 13 candidate target sites in the 3′‐UTR X sequence indicated a uniformly low activity of siRNA constructs against the steady‐state level of replicon. In contrast, the majority of these same siRNAs exhibited high activity against HCV X sequences of either polarity when these targets were presented in the context of a mammalian polymerase II mRNA transcript. Transfection of siRNAs directed against other regions of the replicon revealed differences in the magnitude of inhibitory effects against positive‐strand and negative‐strand target sites. Strand preference of siRNA activity was further demonstrated through the introduction of base‐pair‐destabilizing mutations that promote strand‐specific targeting. The results suggest that the HCV positive‐strand 5′ UTR and coding region are efficiently and directly targeted by siRNA, whereas the 3′ UTR and the entire negative strand are relatively resistant to RNA interference.

Potential role of RNAi in the treatment of HCV infection.

Chronic HCV infection is a leading cause of chronic hepatitis and its sequelae, liver cirrhosis and hepatocellular carcinoma. Current therapeutic options are limited, associated with significant adverse effects and costly. Accordingly, there is strong impetus to develop novel therapeutic strategies that act through alternate mechanisms. RNAi has been widely used for the analysis of gene function and represents a potentially promising approach for the treatment of HCV infection. siRNAs are short RNA duplexes approximately 21 nts long. When introduced into mammalian cells, siRNA can silence specific gene expression. Although efficient suppression of HCV replicon RNA in cell culture has been demonstrated with siRNAs, there is much work to be done to improve delivery, limit off-target effects and minimize development of virus resistance. Here, we review the use of RNAi as a tool to inhibit HCV gene expression and discuss the potential advantages and obstacles for this new potential therapeutic approach against HCV infection.